In humans and a few other primates, the adrenal cortex in the adult individual secretes androgen precursors, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), as well as glucocorticoids and mineral-corticoids. The synthesis of these steroids is performed by a unique cell type of the cortex, the zona reticularis cell. Although the function of these steroids is unknown, the plasma level of DHEAS in the young adult human exceeds that of all other steroid hormones. Equally remarkable is an age-related decline in this level, resulting from the decreased synthesis by the adrenal cortex. This likely results from an age-related loss of ZR cells. They key enzymatic feature of the ZR cell that causes DHEA synthesis is its low expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD). The aims of this proposal are to investigate the molecular and cellular biology of the differentiation of the zones of the human adrenal cortex, specifically the zona reticularis (ZR) and the zona fasciculata (ZF), focusing on the following questions: To clone and characterize a protein that binds to a regulatory region of the type II 3beta-HSD gene and differs in abundance between ZR cells and ZF cells. The bovine adrenal cortex will be used as a source of the protein which will be purified by affinity chromatography; To investigate the possible regulation of ZR cell number by apoptosis. This will be performed using pure isolated ZR cells in culture; To study the behavior of pure ZR cells in a new model of adrenocortical cell and molecular biology using transplantation of adrenal cells into adrenalectomized scid mice. In this model, human ZF cells replace the function of the animals' own adrenal glands. The ability of ZR cells to form functional vascularized tissue in this model will be tested. The emphasis of the proposal is on adrenocortical cell differentiation, using the type II 3beta-HSD gene as the initial focus. More generally, the three specific aims link to explore the control of the differentiation of the ZR cell and its different pattern of gene expression. Its potentially greater susceptibility to apoptosis is one reflection of this pattern of gene expression. Cell transplantation offers the possibility of future testing of molecular mechanisms of zonation under controlled in vivo conditions.